Early onset pediatric epileptic encephalopathies (EEs) such as Dravet Syndrome (DS) are devastating to families because of the high degree of neurodevelopmental compromise, including developmental delay, cognitive decline, and intellectual disability. Most concerning are the severe seizures and high risk of sudden unexpected death in epilepsy (SUDEP). Mutations in voltage-gated Na+ channel (VGSC) ? and ? subunit genes are linked to DS. While the majority of DS cases are linked to SCN1A haploinsufficiency, SCN1B homozygous mutations are also linked to DS (or a DS-like EE). The objective of this work is to understand the mechanism of hyperexcitability in human SCN1B-linked DS/EE. It is hypothesized that the mechanism of Scn1b signaling in neurons involves cell type and subcellular domain specific changes in Na+ current (INa) and K+ current (IK) as well as cell adhesion-mediated effects on neuronal pathfinding and transcriptional regulation of ion channels and transporters. It is proposed that human SCN1B-DS mutations result in defects in neuronal cell-cell communication and ionic currents that are similar to those observed in Scn1b-/- mice. Three Specific Aims are designed to test this hypothesis: 1. To determine whether human SCN1B-linked DS mutations result in loss-of-function in vivo. SCN1B-DS mutations are assumed to result in a functional null phenotype, however, this has not been tested in vivo. A family with an inherited, recessive, SCN1B-DS mutation has donated skin biopsies for induced pluripotent stem cell (iPSC) generation. This mutation, as well as the previously identified SCN1B-R125C human mutation, will be introduced into the mouse Scn1b locus to test homozygous progeny for changes in excitability, neuronal pathfinding, INa, IK, and GABAergic signaling in comparison with Scn1b-/- mice. In parallel, SCN1B-linked human DS iPSC neurons and cerebral organoids will be generated and tested for similar deficits. Gene editing will be used to make isogenic controls, and to generate homozygous null patient-derived neurons to directly compare SCN1B-DS mutant and null cells in the same, isogenic, iPSC line of human neurons. 2. To determine localized changes in INa or IK in Scn1b-/- brain cortical slices. It is possible that the cause of seizures in SCN1B-DS is not disrupted neuronal pathfinding, as previously proposed, but instead neuronal subtype specific changes in INa or IK. Here, a combination of nucleated patch, pulled patches from the AIS, and immunofluorescence staining will be used to determine differential changes in INa and VGSC expression in -/- vs. +/+ cortex. Changes in IK and voltage-gated K+ channel (VGKC) expression will also be tested. 3. To determine whether disruption of Scn1b-mediated neuronal pathfinding plays a role in hyperexcitability in DS. Scn1b-/- mice have neuronal pathfinding defects that precede seizure onset. It was proposed that these defects might lead to the development of seizures. An inducible, pan-neuronal Cre line will be used to delete Scn1b past the critical period of mouse brain development to determine whether seizures and early mortality occur as in Scn1b-/- mice. Here, changes in INa, IK, neuronal patterning, and GABAergic signaling will be investigated following Scn1b deletion at progressive developmental time points. ?1-mediated neurite outgrowth requires trans homophilic ?1-?1 cell adhesion leading to intracellular association of ?1 with ankG in vitro. In a second set of experiments, mutations will be introduced to the mouse Scn1b locus that interrupt ?1-ankG association or ?1 tyrosine phosphorylation to ask whether disruption of the ?1-CAM signaling cascade leads to seizures in vivo. Even though SCN1B- linked DS/EE is a rare disease, this work is important because it will provide new information regarding how deficits in brain development and regulation of ionic currents can synergize to result in hyperexcitability.